The broad and long-term objective of this project is to clarify the nature of infectious vCJD agent that is linked to epidemic BSE: how infectivity spreads, how it can be accurately and rapidly titered, and how this vCJD agent can be identified with certainty. The approaches and models under development here are critical for public health and prevention. They also lay the groundwork for resolving the essential molecular components and fundamental structure of these infectious agents. There are many distinct strains of TSE agents, and vCJD in particular is problematic because of its proven virulence for many species, including humans. It has also been able to transmit from person to person by transfusion from asymptomatic donors, a further reason for concern. The vCJD agent, moreover, retains its characteristics and identity after passage in many different species, and therefore animal models of vCJD are highly relevant for addressing the spread and diagnosis of this infectious agent in the human population. This R21 seeks to establish new experimental animal and tissue culture models to define the vCJD infectious agent more thoroughly, particularly its ability to spread in different tissues where it is not detectable by late pathological markers such as abnormal host prion protein (PrP). We also seek to find faster ways to detect this agent using innovative co-culture strategies. Based on preliminary data at Yale, we have developed the quickest animal model of vCJD to date, and this mouse model can yield critical information for vCJD agent-specific features of spread. Additionally, limited pilot studies here strongly suggest the vCJD agent can infect and replicate in simplified tissue cultures of murine origin. This culture model opens new opportunities for the resolution of the infectious agent that are not possible using complex degenerating brain tissue. This application aims 1) to verify the agent-specificity of both the animal and culture vCJD models, 2) to further develop innovative superinfection tests in culture for strain-specific diagnosis of the vCJD agent, and 3) to use direct culture and superinfection approaches to rapidly reveal infectivity in samples such as blood that are verifiably infectious, but that lack pathological PrP. 4) We will also examine vCJD-infected cultures ultrastructurally to find if they contain the 25nm viruslike particles that have been documented in many TSE-infected, but not control brains. We have discovered comparable viruslike particles in cultures with high titers of scrapie infectivity, and thus we suspect such particles can also aid in the diagnosis, prevention and fundamental understanding of these inevitably lethal infections.